The genome sequence of the tree of heaven, Ailanthus altissima (Mill.) Swingle, 1916

We present a genome assembly from an individual Ailanthus altissima (tree of heaven; Streptophyta; Magnoliopsida; Sapindales; Simaroubaceae). The genome sequence is 939 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules. The mitochondrial and plastid genome assemblies are 661.1 kilobases and 161.1 kilobases long, respectively.


Background
The tree of heaven (Ailanthus altissima, Simaroubaceae) is a deciduous tree native to China and Taiwan, known for its fast growth and attractive foliage.For these reasons, it has been widely planted as an ornamental tree (Figure 1).Ailanthus altissima was first introduced to the UK in 1751 as a garden plant (Hu, 1979), during a period when European fascination with Chinese culture ('Chinoiserie') was at its height.Ailanthus altissima has long been used in Chinese traditional medicine as well as in silk production, as a foodplant for the Ailanthus silk moth (Samia cynthia) (Swingle, 1916).Ailanthus altissima has since become an invasive species across North America, southern South America, South Africa, New Zealand and Europe due to its rapid growth, resistance to pollution, fecundity, vegetative reproduction and use of allelopathic chemicals to stifle competition (Demeter et al., 2021).
The genus name Ailanthus is derived from the Ambonese word 'ailanto', meaning 'heaven-tree', and the specific epithet altissima is Latin for 'tallest', referring to the size of the mature tree.Three varieties of A. altissima have been described -A.altissima var.altissima, native to mainland China, A. altissima var.sutchuenensis (Dode) Rehder and E. H. Wilson, restricted to southern China and A. altissima var.tanakae (Hayata) Kaneh.and Sasaki, endemic to northern Taiwan.Multiple chromosome counts have been reported for A. altissima, ranging from 2n = 80 for arboretum specimens examined at the Royal Botanic Gardens, Kew (Desai, 1960), down to 2n = 64 for samples collected from naturalised populations in mainland Europe (Ivanova et al., 2006), suggesting polyploidy with different cytotypes between individuals.
Here we present the first high-quality genome of A. altissima var.altissima, which we believe will be a useful resource both for those studying the tree in its native range and those aiming to understand what underlies its success as an invasive species.This genome complements existing studies which identified microsatellites for use in population genetic analysis (Dallas et al., 2005;Saina et al., 2021), and those which sequenced the plastome of the species (Saina et al., 2018).Fruitful directions for future work may include investigating the population genetics of invasion, building on previous work showing population bottlenecks in invasive populations of A. altissima (Neophytou et al., 2020).

Genome sequence report
The genome was sequenced from the leaves of an A. altissima specimen (drAilAlti1) collected from the Royal Botanic Gardens, Kew (latitude 51.482, longitude -0.2879).Using flow cytometry, the genome size (1C-value) was estimated as 1.20 pg, equivalent to 1,170 Mb.A total of 45-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 35-fold coverage in 10X Genomics read clouds were generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 17 missing joins or misjoins and removed one haplotypic duplication, reducing the assembly length by 0.11% and the scaffold number by 33.93%, and decreasing the scaffold N50 by 3.45%.
The final assembly has a total length of 939 Mb in 37 sequence scaffolds with a scaffold N50 of 29.4 Mb (Table 1).Most of the assembly sequence (97.72%) was assigned to 31 chromosomal-level scaffolds (Figure 2-Figure 5; Table 2).Several small repetitive scaffolds localise equally well to both chromosome 28 and chromosome 9.They have been placed with chromosome 9, although their placement is uncertain.While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.
Metadata for specimens, spectral estimates, sequencing runs, contaminants and pre-curation assembly statistics can be found at https://links.tol.sanger.ac.uk/species/23810.

Sample acquisition, genome size estimation and nucleic acid extraction
Ailanthus altissima grows as a weed along pavements and in neglected plots in the UK.Leaves from an A. altissima individual (drAilAlti1) were harvested from a plant growing in the pavement near the Jodrell Gate, Royal Botanic Gardens Kew, Surrey, UK (latitude 51.482, longitude -0.2879).The sample was collected and identified by Maarten Christenhusz (Royal Botanic Gardens, Kew).The leaves were preserved by freezing at -80°C.
The genome size was estimated by flow cytometry using the fluorochrome propidium iodide and following the 'one-step' method as outlined in (Pellicer et al., 2021).Specifically for this species, the General Purpose Buffer (GPB) supplemented with 3% PVP and 0.08% (v/v) beta-mercaptoethanol was used for isolation of nuclei (Loureiro et al., 2007), and the internal calibration standard was Petroselinum crispum 'Champion Moss Curled' with an assumed 1C-value of 2,200 Mb (Obermayer et al., 2002).
DNA was extracted at the Tree of Life laboratory, Wellcome Sanger Institute (WSI).The drAilAlti1 sample was weighed and dissected on dry ice with tissue set aside for Hi-C sequencing.Leaf tissue was cryogenically disrupted to a fine powder using Qiagen Plant Magattract, receiving multiple impacts.High molecular weight (HMW) DNA was extracted using the Qiagen MagAttract HMW DNA extraction kit.Low molecular weight DNA was removed from a 20 ng aliquot of extracted DNA using 0.8X AMpure XP purification kit prior to 10X Chromium sequencing; a minimum of 50 ng DNA was submitted for 10X sequencing.HMW DNA was sheared into an average fragment size of 12-20 kb in a Megaruptor 3 system with speed setting 30.Sheared DNA was purified by solid-phase reversible immobilisation using AMPure PB beads with a 1.8× ratio of beads to sample to remove the shorter fragments and concentrate the DNA sample.The concentration of the sheared and purified DNA was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer and Qubit dsDNA High Sensitivity Assay kit.Fragment size distribution was evaluated by running the sample on the FemtoPulse system.
RNA was extracted from leaf tissue of drAilAlti1 in the Tree of Life Laboratory at the WSI using TRIzol, according to the manufacturer's instructions.RNA was then eluted in 50 μL RNAse-free water and its concentration was assessed using a Nanodrop spectrophotometer and Qubit Fluorometer using the Qubit RNA Broad-Range (BR) Assay kit.Analysis of the integrity of the RNA was done using Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.

Sequencing
Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing were performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi), Illumina HiSeq 4000 (RNA-Seq) and Illumina NovaSeq 6000 (10X) instruments.Hi-C data were also generated from material from drAilAlti1 using the Arima v2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020).One round of polishing    , 2022).The mitochondrial and plastid genomes were assembled using MBG (Rautiainen & Marschall, 2021) from PacBio HiFi reads mapping to related genomes.A representative circular sequence was selected for each from the graph based on read coverage.
Table 3 contains a list of relevant software tool versions and sources.Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material

Xin Liu
State Key Laboratory of Agricultural Genomics, BGI (Beijing Genomics Institute)-Shenzhen, Shenzhen, China The manuscript by Rowan et al. described the genome assembly of Ailanthus altissima.They applied PacBio, 10X and HiC sequencing to obtain the sequencing data, and they assembled a chromosome-level genome, with good quality.The method used should be appropriate thus the generated dataset should be valuable for future studies.This type of manuscript is quite straightforward, and I think it is OK for publish as data note.I only have several suggestions for the authors to consider.
With the cytometric evaluation, the genome size was estimated to be 1,170 Mb, which is larger than the assembled genome.Although there would be some acceptable reasons for this, it would be good to have some more information.Have you estimated the genome size using Kmer analysis?If so, it would be good to know the estimation using the sequencing data. 1.
The authors mentioned about the manual curation of the genome assembly to remove some assembly errors.Especially for the haplotypic duplication, it would be better to mention the definition of this type of assembly error.Also, for the contigs corresponding to the second haplotype, it would be more informative to mention the size of the contigs.

2.
Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes

Figure 2 .
Figure 2. Genome assembly of Ailanthus altissima, drAilAlti1.1:metrics.The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 939,218,608 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (38,427,504 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (29,421,908 and 24,184,688 bp, respectively).The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the eudicots_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAMPEX01/dataset/CAMPEX01/snail.

Figure 3 .
Figure 3. Genome assembly of Ailanthus altissima, drAilAlti1.1:GC coverage.BlobToolKit GC-coverage plot.Chromosomes are coloured by phylum.Circles are sized in proportion to chromosome length.Histograms show the distribution of chromosome length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAMPEX01/dataset/CAMPEX01/blob.

Figure 4 .
Figure 4. Genome assembly of Ailanthus altissima, drAilAlti1.1:cumulative sequence.BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all chromosomes.Coloured lines show cumulative lengths of chromosomes assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAMPEX01/dataset/CAMPEX01/cumulative.

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Legality of collection, transfer and use (national and international) Each transfer of samples is further undertaken according to a Research Collaboration Agreement or Material Transfer Agreement entered into by the Darwin Tree of Life Partner, Genome Research Limited (operating as the Wellcome Sanger Institute), and in some circumstances other Darwin Tree of Life collaborators.

Figure 5 .
Figure 5. Genome assembly of Ailanthus altissima, drAilAlti1.1:Hi-C contact map.Hi-C contact map of the drAilAlti1.1 assembly, visualised using HiGlass.Chromosomes are given in order of size from left to right and top to bottom.An interactive version of this figure is available at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=XyRKDNDOTHy19SzkZkKRWw.

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